Project 1. Rapid Automated High-Throughput Radiation Biodosimetry is critical in the context of an Improvised Nuclear Device (IND) or a Radiological Dispersal Device (RDD) event, both for early, dose-based, triage and potentially to predict which exposed individuals are likely to suffer long term illness. The Project 1 rationale is full end-to-end automation - the RABiT (Rapid Automated Biodosimetry Tool) approach - to speed up and improve the precision and accuracy of biodosimetric assays. The proposed research in Project 1 shares the same overall research themes as Project 2 (transcriptomics) and Project 3 (metabolomics): Theme 1: Beyond Simple Dose: Most research in the radiation biodosimetry field has focused on acute external photon irradiation. While this is the simplest exposure case to consider, the potential types of exposure, whether from an IND or a RDD, will vary widely, important factors being A) internal emitter exposures, B) dose protraction, and C) neutron exposure associated with an IND. The goal is to assess how different exposure scenarios modulate the response of radiation biodosimetric markers, and to investigate biomarkers that have specificity for these different exposure scenarios. These factors provide significant opportunities to extend the utility of biodosimetric assays, as well as to understand their mechanistic basis, in what will undoubtedly be very complex radiation fields. Theme 2: Beyond Dose: Towards Individual Radiosensitivity. The goal here is investigation of high- throughput functional assays to predict individual sensitivity to radiation-induced late-occurring injury, in this case pneumonitis. These studies will focus on pneumonitis in non-small cell lung cancer radiotherapy patients, with the goal of correlating this outcome with DNA-damage endpoints measured with the RABiT in ex-vivo irradiated blood samples. Should a significant correlation be found with one of the functional RABiT assay results, this would represent a potential high-throughput screening tool to identify individuals at highest risk of radiation-induced lung disease in a post-IND scenario. Theme 3: Technology Development: Towards Shorter, Faster, Simpler High Throughput Assays: The RABiT is a custom robotically-based fully-automated system specifically tailored for cell-based biodosimetry assays in multi-well plates; since it was developed, commercial robotic high-throughput multipurpose cellular screening platforms have become quite ubiquitous in universities, industry, and clinical testing labs. Thus the goal is to adapt and optimize the RABiT approach / protocols so that they can be used on this new generation of commercial robotic platforms - the RABiT2 approach. To further increase speed, throughput and reliability, the goal is to optimize the assay protocols in terms of accelerated assay time, simplified protocols, simplified image analysis and a shorter time-to-answer, as well as developing new cellular biodosimetric assays that do not require cell culture.